Our long term goal is to understand the mechanisms that control the formation and maintenance of the CNS and PNS myelin, PO, the major protein of PNS myelin, participates in the formation and compaction of the myelin lamellae. PO is esterified with long chain fatty acids, as is PLP, the major protein of CNS myelin. We hypothesize that acylation plays an important role in the process of myelin synthesis and maintenance. The present studies are aimed at determining fundamental aspects of the biology of PO acylation. Our specific aims are: 1. To study the metabolism of the fatty acid bound to PO using a nerve slice system. Double-label pulse and pulse-chase experiments, combined with subcellular fractionation, will be used to identify the membranes in which acylation of PO takes place. We will establish the relationship between acylation and the other PO posttranslational modifications by using inhibitors of protein N-glycosylation and phosphorylation. We will also correlate the extent of PO synthesis and acylation during nerve development to gain insights on the role of this modification. Our preliminary results indicate that acylation occurs only on newly-synthesized but also on a pre- existing PO, suggesting that acylation could also relate to myelin maintenance. 2. To determine the acylation site on the PO molecule. PO will be labeled with [3H] palmitic acid, digested with several proteases and the acyl- peptides isolated and sequenced. 3. To develop a cell-free system using deacylated PO and labeled acyl-CoA as substrates. We will carry out a detailed characterization of the acylating enzyme, localize the subcellular site of cylation unequivocally, isolate the acyltransferase and determine its protein substrate specificity, and determine the developmental and evolutionary changes of this enzyme. The studies proposed in this application will provide basic knowledge on the biology of PO acylation. Moreover, they will contribute to gain insights into normal myelination processes and ultimately to understand the pathophysiology of human peripheral nerve disorders.